Infectious agents such as micro-organisms are typically detected by culturing clinical samples under conditions favourable for the growth of such micro-organisms and monitoring that growth by a number of different techniques including microscopy and detection of more or less specific metabolites of the organisms.
Nucleic acid amplification tests to identify pathogens rapidly and reliably have been implemented in the microbiology laboratory during the last decade. Nucleic acid amplification tests can be used to detect the presence of micro-organisms directly in clinical specimens without culturing.
Initially, identification was accomplished by amplification of a target nucleic acid sequence and detecting of the resulting DNA by visualisation using gel electrophoresis and DNA-binding fluorescent dyes.
Nucleic acid amplification tests revolutionized the world of clinical diagnosis in that they provided an increase in sensitivity and speed of an order of several magnitudes as compared to the classical culture assays.
In general, nucleic acid amplification tests consist of a target specific nucleic acid amplification step and a more or less generic detection step. Herein below follows a brief summary of available amplification techniques and detection platforms.
PCR is currently still the first choice to amplify target sequences and the ability to amplify a wide range of pathogens is dependent on generic, random or multiplex amplification technologies.
In generic PCR tests, only one or two primers pairs are necessary to amplify a target sequence from a range of related pathogens. Regions of conserved nucleotide sequences are required and in general degenerate primer pairs are used.
Several random amplification technologies exist, making use of either random octamers or primers that contain a random 5-8 nucleotide extension at its 3′-end and a defined sequence at its 5′-end. Random amplification is performed in combination with Taq polymerase or isothermal polymerase-based amplification enzymes such as Klenow DNA polymerase or Φ29 DNA polymerase.
Multiplex PCR involves the combination of several primers pairs targeting different sequences in one amplification reaction. Multiplex PCRs require careful optimization to make them comparably sensitive and specific as single pair amplification reactions.
Multiplex Ligation-dependent Probe Amplification (MLPA) technology (Schouten et al; WO 01/61033, Schouten et al., Nucl. Acids Res. 2002, vol 30 No 12; e57) is a multiplex PCR method capable of amplifying different targets simultaneously. In MLPA two oligonucleotides that hybridise immediately adjacent to each other on target DNA are added in the same reaction. One of the oligonucleotides is synthetic and has a size of 40-60 nucleotides (nt) whereas the other oligonucleotide has a size ranging from 100 up to 400 nt and requires a cloning step in an M13 vector to finally generate single stranded probe DNA. MLPA consists of three steps: first an annealing step to hybridise the probes to their target region, secondly a ligation step to covalently link the two probes together and thirdly the final PCR to get an exponential amplification of the target regions using only two universal primers.
Currently, target specific multiplex PCR amplification is the standard method for pathogen detection assays.
Because of the extreme sensitivity of nucleic acid amplification tests, care must be taken to avoid contamination in these tests. Detection of amplified nucleic acids was originally performed by size determination using gel electrophoresis and intercalating DNA dyes. A first step in minimizing contamination was taken when the amplification and detection steps were combined into one step. As a result, post-amplification handling steps were eliminated, thereby adding to the reliability of the assay. Such assays are often referred to as a closed system. Closed system amplification technologies such as real time PCR (Ratcliff et al. Curr Issues Mol Biol. 2007, 9(2): 87-102) and NASBA (Loens et al., J Clin Microbiol. 2006, 44(4); 1241-1244) and LAMP (Saito R et al., J Med Microbiol. 2005, 54; 1037-41) have been developed and use intercalating fluorescent dyes or fluorescent labelled probes. The isothermal LAMP technology allows real time detection by spectrophotometric analysis using a real-time turbidimeter. Currently, most of these assays are organism-specific and useful only when a particular pathogen is suspected. This limits the scope of these assays considerably.
However, clinical symptoms are only rarely attributable to a single pathogen. Hence, there is a need in the art for assays that allow the simultaneous identification and differentiation of multiple agents. Such multi-parameter assays enable the clinician to come to a faster and better therapy and contribute to improved clinical management and public health.
For this reason technologies have been developed for the purpose of testing simultaneously for more than one organism. One of such technologies is multiplex real time PCR. At present, however, only five colour oligo-probe multiplexing is possible of which one colour is ideally set aside for an internal control to monitor inhibition and perhaps even acts as a co-amplified competitor (Molenkamp et al. J. Virol Methods, 2007, 141: 205-211). This considerably limits the amount of pathogens that may be tested simultaneously.
An example of an area where it is particularly desirable to have a quick, reliable and specific multiplex assay for several pathogens at once, is the area of respiratory tract infections.
Acute respiratory tract infection is the most widespread type of acute infection in adults and children. The number of pathogens involved is numerous. Respiratory tract infections (RTI) are commonly divided into upper respiratory tract infections (URTI) and lower respiratory tract infections (LRTI). The URTI include rhinorrhea, conjunctivitis, pharyngitis, otis media and sinusitis and LRTI include pneumoniae, brochiolitis and bronchitis. Both viruses and bacteria cause acute RTI, and the number of causative agents is large as well as diverse.
Non-typical viruses and bacteria involved in RTI include influenza virus A and B (InfA and B), parainfluenza virus 1, 2, 3 and 4 (PIV-1, -2, -3 and 4), respiratory syncytial virus A and B (RSVA and B), rhinovirus, coronavirus 229E, OC43 and NL63 (Cor-229E, -OC43 and NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), human metapneumovirus (hMPV), adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila and Bordetella pertussis. Many of these infections are indistinguishable by clinical features alone and require rapid laboratory tests for optimal patient management and infection control.
Viral culture is still the gold standard for laboratory diagnosis of respiratory viruses. However, viral culture is relatively slow and therefore routine diagnosis is sub optimal. Although rapid antigen detection tests are available for some of these viruses, these tests have shown to be less sensitive and less specific than viral culturing. Currently, there is a desperate need for a sensitive and specific method for the simultaneous detection of respiratory viruses in a multiplex format. It would be very advantageous to be able to detect two or more targets in a single reaction as this would provide distinct advantages in clinical diagnostics. It would simplify the assay, increase the throughput, minimize the consumption of clinical specimen and in particular multiplex assays would be more cost-effective than monoplex assays.
To differentially detect respiratory viruses in clinical specimens the following detection platforms have been used:
(i) Gel Electrophoresis.
Using agarose gel electrophoresis as detection device, several nested multiplex reverse transcriptase (RT)-PCR assays have been developed using three or four primer pairs. Osiowy et al., (J. Clin. Microbiol. 1998, 36; 3149-3154) used five primers pairs that amplified RNA from respiratory syncytial virus A and B, parainfluenza virus 1, 2 and 3 and adenovirus types 1 to 7. The PCR products varied in size from 84 up to 348 base pairs. Compared to direct immunofluorescence (DIF) assays or indirect immunofluorescence (IIF) assays a sensitivity value of 91% and a specificity value of 87% was obtained for this multiplex RT-PCR approach.
Coiras et al. (J. Med. Virology, 2004, 72; 484-495) using the same approach, were able to simultaneously detect 14 respiratory viruses in two multiplex RT nested PCR assays. They included coronavirus 229E and OC43, rhinovirus, enterovirus, parainfluenza virus 4 and an internal control but omitted adenovirus 1 to 7. The assay was evaluated on nose and throat swaps and nasopharyngeal aspirates from infants below two years of age. It appeared that the multiplex assay was more sensitive than conventional viral culture and immunofluorescence assays, with the advantage that all viruses can be tested at the same time and with a single technique. In addition, in 9.5% of the samples a double infection was found.
Erdman et al. (J. Clin. Microbiol. 2003, 41; 4298-4303) recently developed a RT-PCR assay against 6 common respiratory viruses based on automated fluorescent capillary electrophoresis and Genescan software for detection of respiratory syncytial virus A and B, parainfluenza virus 1, 2 and 3 and influenza virus A and B. An one-step RT-PCR reaction was performed using primers of which the positive strand primer of each primer set was 5′ end labelled with the fluorescent dye 6-carboxyfluorescein (6-FAM). Overall, this RT-PCR assay was positive in 92% of the samples that were also positive by culture or DIF staining.
The above references are examples of techniques wherein a large number of samples is analysed using gel-electrophoresis. Disadvantages of gel electrophoresis as a detection technique are that it is laborious and time consuming and therefore rather costly. Furthermore, the risk of cross contamination is enlarged as each sample has to be opened after the PCR for analysis.
(ii) Secondary Enzyme Hybridisation.
In this approach multiplex PCR assays are combined with an enzyme-linked immunosorbent assay (ELISA).
Detection of the multiplex PCR products is performed by microwell hybridisation analysis in streptavidin-coated wells of a microtiter plate. Biotinylated capture probes specific for the amplified target sequences are added and a peroxidase labelled hybridisation reaction is performed. Subsequently, the optical density is measured by a reader/spectrophotometer. Samples are classified as PCR positive or negative depending on the cut-off optical density value.
Multiplex RT-PCR enzyme hybridisation assays for rapid identification of seven or nine micro-organisms causing a respiratory tract infection have been developed and validated in comparison to the gold standard. The commercially available Hexaplex assay (Prodesse, Inc., Milwaukee, Wis.) (Fan et al, Clin. Infect. Dis. 1998, 26; 1397-1402, Kehl et al., J. Clin. Microbiol. 2001, 39; 1696-1701 and Liolios et al., J. Clin. Microbiol. 2001, 39; 2779-2783) is directed against parainfluenza virus 1, 2 and 3, respiratory syncytial virus A and B and influenza virus A and B whereas the nineplex assay contained the same RNA viruses minus parainfluenza 2 and respiratory syncytial virus A and B were combined in one primer pair but including enterovirus, adenovirus and two bacteria Mycoplasma pneumoniae and Chlamydia pneumoniae (Grondahl et al., J. Clin. Microbiol. 1997, 37; 1-7 and Puppe et al., J. Clin. Virol. 2003, 30; 165-174). The analytical sensitivity of the Hexaplex assay has been shown to be 100-140 copies/ml depending on the virus, whereas the nineplex assay was less sensitive compared to culture for respiratory syncytial virus and parainfluenza virus 1 and more sensitive for parainfluenza virus 3, influenza virus A and B, adenovirus and enterovirus. The analytical sensitivity was measured on serial dilutions of viral culture supernatants. The sensitivity and specificity of the nineplex and hexaplex on clinical specimens varied between the RNA viruses and was found to be between 86% -100% for sensitivity and 80%-100% for specificity. Both assays were compared with monoplex RT-PCR ELISA and other monoplex RT-PCR tests and were approximately of the same quality. Although the ELISA based assays allow highly multiplex analyses, they posses the same disadvantages as the gel electrophoresis based assays. The assays are laborious and time consuming and the reaction vessel has to be opened after PCR, thereby increasing the risk of cross contamination.
(iii) Measuring Emission Using Different Fluorescent Dyes.
Fluorescence reporter systems such as real time PCR have been introduced in the diagnostic laboratory recently. Real Time PCR combines DNA amplification with detection of the products in a single tube. Detection is based on changes in fluorescence proportional to the increase in product. Real Time PCR capacity to simultaneously detect multiple targets is limited to the number of fluorescent emission peaks that can be unequivocally resolved. At present, only four colour oligoprobe multiplexing is possible of which one colour is ideally set aside for an internal control to monitor inhibition and perhaps even acts as a co-amplified competitor.
Many monoplex or duplex real-time PCR assays against respiratory pathogens have been developed either being home brew based (van Elden et al., J. Clin. Microbiol. 2001, 39; 196-200, Hu et al, J. Clin. Microbiol. 2003, 41; 149-154) or commercially available assays (Prodesse, Inc., Milwaukee, Wis.). One of the first multiplex real-time PCR assays directed against respiratory viruses was developed by Templeton et al. (J. Clin. Microbiol. 2004, 42; 1564-1569). A real-time multiplex PCR assay was developed for the detection of 7 respiratory RNA viruses (influenza virus A and B, respiratory syncytial virus, parainfluenza virus 1, 2, 3 and 4) in a two-tube multiplex reaction. Each assay was initially set up as a monoplex assay and then combined in two multiplex assays: one comprises influenza virus A and B and respiratory syncytial virus whereas as the other one comprises parainfluenza virus 1, 2, 3 and 4 with both assays having the same PCR protocol so they could be run in parallel. No non-specific reactions or any inter-assay cross-amplification was observed and only the correct virus was amplified by the two multiplex reactions. Clinical evaluation was performed by viral culture and confirmed by IF and multiplex PCR on the same samples. Viral culture resulted in 19% positive samples whereas multiplex resulted in 24% positives. The multiplex PCR-positive specimens included all the samples that were positive by viral culture and additional ones. The additional ones were tested by a second PCR-assay and it could be shown that that these samples were true positives.
For simultaneous detection of 12 respiratory RNA viruses by real-time PCR, Gunson et al. (J. Clin. Virol. 2005, 33; 341-344) developed four triplex reactions: (i) influenza virus A and B and human metapneumovirus, (ii) respiratory syncytial virus A and B and rhinovirus, (iii) parainfluenza virus 1, 2 and 3 and (iv) coronavirus 229E, OC43 and NL63. These 4 assays cover almost the complete set of respiratory RNA viruses and implementation of these assays was said to improve patient management, infections control procedures and the effectiveness of surveillance systems. The real time PCR assays allow analysis without any post PCR handling of the sample. This diminishes the risk of cross contamination and requires no extra handling time. However, the complexity of the current assays is limited to a maximum of four probes per reaction. Moreover, complex analyses require more reactions thereby increasing the costs.
(iv) Microarrays Consisting of Oligonucleotides or PCR Amplicons Immobilized on a Solid Surface.
Microarrays for diagnostic purposes require either (a) genome specific probes to capture the unknown target sequences or (b) generic zipcodes present in the amplified target sequence and thereby reveal the presence of that pathogen in a clinical specimen. Hybridisation between the bound probe and target sequence in the sample is revealed by scanning or imaging the array surface.
DNA microarrays offer the possibility for highly parallel viral screening to simultaneously detect hundreds of viruses. Related viral serotypes could be distinguished by the unique pattern of hybridisation generated by each virus. High density arrays are able to discriminate between ten thousand different targets whereas low density arrays of up to a few hundred targets are more appropriate in clinical diagnostics. The first array for use in diagnostic virology was constructed by Wang at al. (Proc. Natl. Acad. Sci. USA 99; 15678-15692). They initially constructed a microarray of 1600 unique 70-mer oligonucleotide probes designed from about 140 viral genome sequences of which the respiratory tract pathogens were of major concern. The viral RNA was amplified using a randomly labelled PCR procedure and the array was validated with nasal lavage specimens from patients with common colds. The array detected respiratory pathogens containing as few as 100 infectious particles. The data were confirmed with RT-PCR using specific PCR primers. Cross hybridisation was only observed to its close viral relatives.
Low density arrays have been constructed for detection, typing and sub-typing of Influenza (Kessler et al., J. Clin. Microbiol. 2004, 42; 2173-2185) and acute respiratory disease-associated adeno viruses (Lin et al., J. Clin Microbiol. 2005, 42; 3232-3239). The Influenza chip was shown to detect as few as 1×102 to 5×102 influenza virus particles whereas the sensitivity of the adeno microarray was 103 genomic copies when clinical samples were analysed directly. Multiplex as well as random amplification procedures were used.
A very new development in respiratory tract pathogen identification is the use of re-sequencing microarrays (Lin et al., Genome Res., 2006, 16:527-535, and Wang et al, Bioinformatics 2006 22(19):2413-2420; doi:10.1093/bioinformatics/bt1396). The exponentially increasing availability of microbial sequences makes it possible to use direct sequencing for routine pathogen diagnostics. However, this requires that pathogen sequence information be rapidly obtained. Resequencing microarrays use tiled sets of 105 to 106 probes of either 25-mers or 29-mers, containing one perfectly matched and three mismatched probes per base for both strands of target genes. A custom designed Affymetrix re-sequencing Respiratory Pathogen Microarray (RPM v.1) has been disclosed. This RPM v.1 array harbours 14 viral and bacterial species. A random amplification protocol was used and in both studies identification not only at the species level but also at the strain level was obtained. This is of particular interest for surveillance of epidemic outbreaks. The development of a second RPM chip (v.2) has already been initiated including 54 bacterial and viral species. However, the sensitivity and assay speed has to be improved to provide a diagnostic platform for pathogen detection.
The immediate precursor of a DNA array suitable in clinical diagnostics was the reverse hybridisation line probe or blot. Line probe/blot assays have been described for mutation detections and for genotyping and are also commercially available (line probe assay (LiPA) from Innogenetics, Belgium). Generally, a generic amplification technology is used but up to now no studies have been published of line probe blots against respiratory viral pathogens. The microarray based assays allow highly complex analyses. However, they require specialized equipment and extensive post PCR handling.
(v) Beads or Microspheres Systems
In these products, detection is performed by a flow cytometer. In such a system microspheres are internally dyed with two spectrally distinct fluorochromes. Using precise amounts of each of these fluorochromes, an array is created consisting of 100 different microspheres sets with specific spectral properties. Due to this different spectral property, microspheres can be combined, allowing up to 100 different targets to be measured simultaneously in a single reaction. For nucleic acid detection using microspheres, direct hybridisation of a labelled PCR amplified target DNA to microspheres bearing oligonucleotide capture probes products specific for each target sequence are used. Detection is performed by two lasers, one to identify the distinct bead set and the other one to determine the specific target sequence.
Microsphere-based suspension array technologies, such as the Luminex® xMAP™ system, offer a new platform for high throughput multiplex nucleic acid testing. Compared to planar microarrays, they have the benefits of faster hybridisation kinetics and more flexibility in array preparation. Recently, a novel microsphere-based universal array platform, called the Tag-It™ platform has been developed and used for detection and differentiation of 19 respiratory viruses. The Tag-It™ array platform features universal, minimally cross-hybridizing tags for capturing the reaction products by hybridisation onto complementary anti-tag coupled microspheres. The respiratory viral panel on the Luminex platform was developed by TM Biosciences (Toronto, Canada) and validated on nasopharyngeal swabs and aspirates. An overall sensitivity of 96.1% was obtained and data were confirmed by monoplex PCR and DFA. In addition 12 double (out of 294 specimen samples) infections were detected. As with the microarray based assays, these assays allow highly complex analyses but require specialized equipment and extensive post PCR handling.
(vi) Mass Spectrometry Systems
These are assays wherein tags are released by UV irradiation and subsequently analyzed by a mass spectrometer. Oligonucleotide primers, designed against conserved regions of the pathogen, are synthesized with a 5′ C6 spacer and aminohexyl modification and covalently conjugated by a photo-cleavable link to (Masscode) tags. A library of 64 different tags has been established. Forward and reverse primers in individual primer sets are labelled with distinct molecular tags. Amplification of a particular pathogen target results in a dual signal in a mass spectrometer that allows assessment of specificity.
Mass spectrometry is a homogeneous solution assay format that allows for simultaneous detection of multiple nucleic acid sequences in a single reaction thereby reducing time, labour and cost as compared to single-reaction-based detection platforms. A new class of molecular labels, called cleavable mass spectrometry tags (CMSTs) has been developed for simultaneous data acquisition. One application of CMST technology is termed Masscode (Qiagen, Hilden, Germany) and is used for differential detections of respiratory pathogens. The general structure of CMSTs is highly modular and includes a photolabile linker, a mass spectrometry sensitivity enhancer and a variable mass unit all connected through a scaffold constructed around a central lysine residue. CMSTs are attached to the 5′-end of the oligonucleotide of the PCR primer through a photo-cleavable linker. The combination of the enhancer and the variable mass unit specify the final mass of each individual CMST. Currently, a library of 64 distinct Masscode tags has been developed and a variety of mass spectrometry ionization methods can be applied. A great advantage of detection by mass spectrometry is the speed. Analysis takes only a few seconds.
Briese et al. (Emerg. Infect. Dis., 2005, 11; 310-313) developed a diagnostic assay comprising of 30 gene targets that represented 22 respiratory pathogens. Nucleic acid from banked sputum, nasal swabs and pulmonary washes was tested and compared to virus isolation and conventional nested RT-PCR. Consistent results were obtained. The detection threshold was between 100-500 copies per sample. Mass spectrometry is a very fast technique enabling highly complex analyses. However, this application requires specialized and expensive hardware which, at the moment, is not common on a standard microbiology laboratory.
The above described multiparameter approaches to identify and differentiate the causative agents of a RTI are a great step forward but still have limitations. They take either too much time (>10 hours), require a lot of hands-on time, have a limited multi-parameter character and/or need expensive equipment or large set-up costs to perform the tests.